Artificial transcription factors

ABSTRACT

Transcription regulating polypeptides that contain a plurality of DNA binding domains are provided. The polypeptides optionally contain one or more transcription regulating domains. Polynucleotides that encode the polypeptides and use of the polypeptides and polynucleotides are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent is a continuation-in-part of U.S. Provisional PatentApplication Ser. No. 60/388,055, filed Jun. 11, 2002, the disclosure ofwhich is incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The field of this invention is gene transcription. More particularly,this invention provides a gene transcription regulating polypeptide thatcontains a plurality of DNA binding domains directed to different targetnucleotide sequences within one or more genes.

BACKGROUND OF THE INVENTION

The construction of artificial transcription factors has been of greatinterest in the past years. Gene expression can be specificallyregulated by polydactyl zinc finger proteins fused to regulatory domains(See, e.g., U.S. Pat. Nos. 6,242,568; 6,140,466; and 6,140,081, thedisclosures of which are incorporated herein by reference).

Zinc finger domains of the Cys₂-His₂ family have been most promising forthe construction of artificial transcription factors due to theirmodular structure. Each domain consists of approximately 30 amino acidsand folds into a ββα structure stabilized by hydrophobic interactionsand chelation of a zinc ion by the conserved Cys₂-His₂ residues. Todate, the best characterized protein of this family of zinc fingerproteins is the mouse transcription factor Zif 268 [Pavletich et al.,(1991) Science 252(5007), 809-817; Elrod-Erickson et al., (1996)Structure 4(10), 1171-1180]. The analysis of the Zif268/DNA complexsuggested that DNA binding is predominantly achieved by the interactionof amino acid residues of the α-helix in position −1, 3, and 6 with the3′, middle, and 5′ nucleotide of a 3 bp DNA subsite, respectively.Positions 1, 2 and 5 have been shown to make direct or water-mediatedcontacts with the phosphate backbone of the DNA. Leucine is usuallyfound in position 4 and packs into the hydrophobic core of the domain.Position 2 of the α-helix has been shown to interact with other helixresidues and, in addition, can make contact to a nucleotide outside the3 bp subsite [Pavletich et al., (1991) Science 252(5007), 809-817;Elrod-Erickson et al., (1996) Structure 4(10), 1171-1180; Isalan, M. etal., (1997) Proc Natl Acad Sci U S A 94(11), 5617-5621].

Zinc finger DNA binding domains can be assembled into zinc fingerproteins recognizing extended 18 bp DNA sequences which are uniquewithin the human or any other genome. In addition, these proteinsfunction as transcription factors and are capable of altering geneexpression when fused to regulatory domains and can even be madehormone-dependent by fusion to ligand-binding domains of nuclear hormonereceptors. To date, however, polypeptides containing one or more zincfinger binding domains target a single gene or contain a singletranscription regulating domain. There is a need in the art, therefore,for transcription regulating polypeptides that can be used to targetmore than one gene or contain more than one transcription regulatingdomain.

The present disclosure provides polypeptides that contain a plurality ofDNA binding domains and one or more transcription regulating domains.Such polypeptides can be used to regulate transcription of more than onetarget gene or to enhance the activation or repression of single genes.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a non-naturally occurring artificialtranscription factor polypeptide comprising a plurality of DNA bindingdomains (DNB) operatively linked to each other. The DNA binding domainseach bind independently to a same or different nucleotide sequence. Thepolypeptide can further contain one or more transcription regulatingdomains, each of which is operatively linked to one of the DNA bindingdomains.

The different nucleotide sequences are located in a transcriptionalcontrol region of the same gene or different genes. Where the nucleotidesequences are located in transcriptional control regions of a singlegene, such nucleotide sequences are separated from each other by atleast 10 base pairs. The polypeptide contains two or more DNBs. In oneembodiment, the polypeptide contains two or three DNA binding domains.Each DNA binding domain preferably contains from 3 to 6 zinc fingerpeptides and, more preferably 6 zinc finger peptides.

The DNA binding domains are preferably operatively linked to each otherwith an amino acid residue sequence of from 5 to 50 amino acid residues,preferably from 5 to 40 amino acid residues, more preferably from 5 to30 amino acid residues and, even more preferably from 5 to 15 amino acidresidues.

In another aspect, the present invention provides a polynucleotide thatencodes a polypeptide of this invention, an expression vector thatcontains such a polynucleotide and a cell transformed with such apolynucleotide or expression vector.

In yet another aspect, the present invention provides processes forregulating gene transcription. In one embodiment, a present method isdirected to simultaneously regulating transcription of a plurality ofDNA target genes in a cell. Such a method comprises the steps oftransforming the cell with a polynucleotide that encodes a polypeptidehaving a plurality of operatively linked DNA binding domains, each ofwhich DNA binding domains specifically binds to a nucleotide sequence ina transcriptional control region of different DNA target genes andmaintaining the cell under conditions and for a period of timesufficient for expression of the polypeptide. In a second embodiment, amethod is directed to regulating transcription of a single gene. Such amethod comprises the steps of transforming the cell with apolynucleotide that encodes a polypeptide having a plurality ofoperatively linked DNA binding domains, each of which DNA bindingdomains specifically binds to a different nucleotide sequence in atranscriptional control region of the DNA target gene and maintainingthe cell under conditions and for a period of time sufficient forexpression of the polypeptide. Preferably, a method of this inventionuses a polypeptide that also contains one or more transcriptionregulating domains.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings that form a portion of the specification,

FIG. 1 shows a schematic representation of a transcription factorpolypeptide of this invention. DNB represents a DNA binding domain. N isfrom 1 to 10. ˜Represents an amino acid residue linker.

FIG. 2 shows exemplary arrangements of DNBs and repressor (SKD) oractivation (VP64) transcription regulating domains assembled out of twoDNBs connected by a flexible linker.

DETAILED DESCRIPTION OF THE INVENTION

I. The Invention

The present invention provides non-naturally occurring transcriptionfactor polypeptides useful for regulating gene transcription,polynucleotides that encode such polypeptides and the use of suchpolypeptides and polynucleotides in regulating gene transcription.

II. Polypeptides

The present invention provides non-naturally occurring polypeptides thatcontain a plurality of DNA binding domains (DNB), which binding domainsare derived from zinc finger DNA binding peptides (See FIG. 1). Apolypeptide of this invention is non-naturally occurring. As usedherein, the term “non-naturally occurring” means, for example, one ormore of the following: (a) a polypeptide comprised of a non-naturallyoccurring amino acid sequence; (b) a polypeptide having a non-naturallyoccurring secondary structure not associated with the polypeptide as itoccurs in nature; (c) a polypeptide that includes one or more aminoacids not normally associated with the species of organism in which thatpolypeptide occurs in nature; (d) a polypeptide that includes astereoisomer of one or more of the amino acids comprising thepolypeptide, which stereoisomer is not associated with the polypeptideas it occurs in nature; (e) a polypeptide that includes one or morechemical moieties other than one of the natural amino acids; or (f) anisolated portion of a naturally occurring amino acid sequence (e.g., atruncated sequence). A polypeptide of this invention exists in anisolated form and purified to be substantially free of contaminatingsubstances. A polypeptide can be synthetic in nature. That is, thepolypeptide is isolated and purified from natural sources or made denovo using techniques well known in the art. A polypeptide of thisinvention can be made using a variety of standard techniques well knownin the art.

Amino acid residues of polypeptides are expressed herein using thestandard 1 or 3-letter codes (See Table 1, below). TABLE 1 3 Letter 1Letter Amino Acid Code Code Alanine Ala A Cysteine Cys C Aspartic AcidAsp D Glutamic Acid Glu E Phenylalanine Phe F Glycine Gly G HistidineHis H Isoleucine Ile I Lysine Lys K Leucine Leu L Methionine Met MAsparagine Asn N Proline Pro P Glutamine Gln Q Arginine Arg R Serine SerS Threonine Thr T Valine Val V Tryptophan Trp W Tyrosine Tyr Y StopCodons Z

In certain embodiments, a polypeptide variant comprises a conservativelysubstituted amino acid residue. It is preferred that each amino acidsubstitution is made by substituting the amino acid of interest with anamino acid from a group of similar amino acid(s) as listed in the Table2, below. (See Biochemistry, 3rd Edition, Stryer, Freeman Publisher(1988) pages 16-40, incorporated herein by reference).

Referring to the Table 2, for example, in certain embodiments, a G aminoacid residue in a desired polypeptide is substituted with an A, V, L, orI. In another example, an N residue in a desired polypeptide issubstituted with a D, E, or Q. It is generally preferred that the firstamino acid (or codon in the underlying polynucleotide) of an openreading frame is methionine. TABLE 2 Preferred Amino Acid Grouping forConservative Substitution Conservative Side Chain Characteristic AminoAcid Groups Aliphatic G, A, V, L, I Aliphatic with secondary amino groupP Aromatic F, Y, W Sulfur containing C, M Aliphatic hydroxyl S, T BasicK, R, H Acidic D, E, N, Q

A DNA binding domain of an instant polypeptide is derived or isolatedfrom zinc finger DNA binding peptides, which peptides are well known inthe art. Preferably, the zinc finger DNA binding peptide is derived froma Cys₂-His₂ type zinc finger. A zinc finger DNA binding peptidederivative can be derived or produced from a wild type zinc fingerprotein by truncation or expansion, or as a variant of a wildtype-derived peptide by a process of site directed mutagenesis, or by acombination of the procedures (See e.g., U.S. Pat. Nos. 6,242,568;6,140,466; and 6,140,081, the disclosures of which are incorporatedherein by reference). The term “truncated” refers to a zincfinger-nucleotide binding polypeptide that contains less that the fullnumber of zinc fingers found in the native zinc finger binding proteinor that has been deleted of non-desired sequences. For example,truncation of the zinc finger-nucleotide binding protein TFIIIA, whichnaturally contains nine zinc fingers, might be a polypeptide with onlyzinc fingers one through three. Expansion refers to a zinc fingerpolypeptide to which additional zinc finger modules have been added. Forexample, TFIIIA may be extended to 12 fingers by adding 3 zinc fingerdomains.

In addition, a truncated zinc finger-nucleotide binding polypeptide mayinclude zinc finger modules from more than one wild type polypeptide,thus resulting in a “hybrid” zinc finger-nucleotide binding polypeptide.The term “mutagenized” refers to a zinc finger derived-nucleotidebinding polypeptide that has been obtained by performing any of theknown methods for accomplishing random or site-directed mutagenesis ofthe DNA encoding the protein. For instance, in TFIIIA, mutagenesis canbe performed to replace nonconserved residues in one or more of therepeats of the consensus sequence. Truncated zinc finger-nucleotidebinding proteins can also be mutagenized. Examples of known zincfinger-nucleotide binding polypeptides that can be truncated, expanded,and/or mutagenized according to the present invention in order toinhibit the function of a nucleotide sequence containing a zincfinger-nucleotide binding motif includes TFIIIA and zif268. Other zincfinger-nucleotide binding proteins will be known to those of skill inthe art.

A polypeptide of this invention comprises a plurality of DNA bindingdomains. Preferably, the polypeptide contains from 2 to 10 such domains,more preferably from 2 to 5 such domains and, most preferably, 2 or 3such domains. The DNA binding domains are operatively linked to eachother. By “operatively linked” is meant that the structure and functionof each DNA binding domain is unaffected by the linking of any othersuch domain. In one embodiment, the DNA binding domains are directlylinked or bonded together via well known peptide linkages. In anotherembodiment, the DNA binding domains are operatively linked using apeptide linker containing from 5 to 50 amino acid residues. Preferably,the linker contains from 5 to 40 amino acid residues, more preferablyfrom 5 to 30 amino acid residues and, even more preferably from 5 to 15amino acid residues. The linkers are preferably flexible. Exemplary suchlinkers are set forth below. (SEQ ID NO:1) Linker 1: TGEKP (SEQ ID NO:2)Linker 2: PGGGGSGGGGTGSSRSSSTGEKP (SEQ ID NO:3) Linker 3:PGSSGGGGSGGGGGGSTGGGSGGGGTGSSRSSSTGEKP (SEQ ID NO:4) Linker 4:TGGGGSGGGGTGEKP

Where a transcription factor polypeptide of this invention contains 2DNA binding domains, a single linker operatively links those domains.Where more than two DNA binding domains are present, a linker is used tooperatively link each binding domain. In such an embodiment, the same ordifferent linker can be employed at each linking location.

DNA binding domains used in the present transcription factors can benaturally-occurring or non-naturally occurring. Naturally-occurring zincfinger DNA binding domains are well known in the art. In a preferredembodiment, at least one DNA binding domain of a present transcriptionfactor is non-naturally occurring. Each of the DNA binding domains ispreferably designed and made to specifically bind nucleotide targetsequences corresponding to the formula 5′-NNN-3′, where N is anynucleotide (i.e., A, C, G or T). Such DNA binding domains are well knownin the art (See. e.g. U.S. Pat. Nos. 6,242,568, 6,140,466 and 6,140,081,the disclosures of which are incorporated herein by reference). A zincfinger DNA binding peptide of this invention comprises a unique heptamer(contiguous sequence of 7 amino acid residues) within the α-helicaldomain of the peptide, which heptameric sequence determines bindingspecificity to a target nucleotide. That heptameric sequence can belocated anywhere within the a-helical domain but it is preferred thatthe heptamer extend from position −1 to position 6 as the residues areconventionally numbered in the art. A peptide can include any β-sheetand framework sequences known in the art to function as part of a zincfinger peptide.

Previously we reported the characterization of 16 zinc finger domainsspecifically recognizing each of the 5′-GNN-3′ type of DNA sequences,that were isolated by phage display selections based on C7, a variant ofthe mouse transcription factor Zif268 and refined by site-directedmutagenesis [U.S. Pat. No. 6,140,081, the disclosure of which isincorporated herein by reference]. Briefly, phage display libraries ofzinc finger proteins were created and selected under conditions thatfavored enrichment of sequence specific proteins. Zinc finger domainsrecognizing a number of sequences required refinement by site-directedmutagenesis that was guided by both phage selection data and structuralinformation. A similar system has been employed to identify domains thatrecognize the 5′-TNN-3′ type of DNA sequences.

To extend the availability of zinc finger domains for the constructionof artificial transcription factors, domains specifically recognizingthe 5′-ANN-3′ and 5′-CNN-3′ types of DNA sequences were selected.Briefly, the helix TSG-N-LVR (SEQ ID NO: 5), previously characterized infinger 2 position to bind with high specificity to the triplet5′-GAT-3′, containing finger 1 and 2 of C7 and the 5′-GAT-3′-recognitionhelix in finger-3 position, was analyzed for DNA-binding specificity ontargets with different finger-2 subsites by multi-target ELISA incomparison with the original C7 protein [See, e.g., Dreier, B. et al.: JBiol Chem Aug. 3, 2001; 276(31):29466-78].

A polypeptide of this invention can further comprise one or moretranscription regulating domains. A transcription regulating domain canbe an activation domain or a repression domain, as is well known in theart. An exemplary repression domain peptide is the ERF repressor domain(ERD), (Sgouras, D. N., Athanasiou, M. A., Beal, G. J., Jr., Fisher, R.J., Blair, D. G. & Mavrothalassitis, G. J. (1995) EMBO J. 14,4781-4793), defined by amino acids 473 to 530 of the ets2 repressorfactor (ERF). This domain mediates the antagonistic effect of ERF on theactivity of transcription factors of the ets family. A second repressorprotein is prepared using the Krüppel-associated box (KRAB) domain(Margolin, J. F., Friedman, J. R., Meyer, W., K.-H., Vissing, H.,Thiesen, H.-J. & Rauscher III, F. J. (1994) Proc. Natl. Acad. Sci. USA91, 4509-4513). This repressor domain is commonly found at theN-terminus of zinc finger proteins and presumably exerts its repressiveactivity on TATA-dependent transcription in a distance- andorientation-independent manner (Pengue, G. & Lania, L. (1996) Proc.Natl. Acad. Sci. USA 93, 1015-1020), by interacting with the RING fingerprotein KAP-1 (Friedman, J. R., Fredericks, W. J., Jensen, D. E.,Speicher, D. W., Huang, X.-P., Neilson, E. G. & Rauscher III, F. J.(1996) Genes & Dev. 10, 2067-2078). We utilized the KRAB domain foundbetween amino acids 1 and 97 of the zinc finger protein KOX1 (Margolin,J. F., Friedman, J. R., Meyer, W., K.-H., Vissing, H., Thiesen, H.-J. &Rauscher III, F. J. (1994) Proc. Natl. Acad. Sci. USA 91, 4509-4513). Inthis case an N-terminal fusion with a zinc-finger polypeptide isconstructed. Finally, to explore the utility of histone deacetylationfor repression, amino acids 1 to 36 of the Mad mSIN3 interaction domain(SID) are fused to the N-terminus of the zinc finger protein (Ayer, D.E., Laherty, C. D., Lawrence, Q. A., Armstrong, A. P. & Eisenman, R. N.(1996) Mol. Cell. Biol. 16, 5772-5781). This small domain is found atthe N-terminus of the transcription factor Mad and is responsible formediating its transcriptional repression by interacting with mSIN3,which in turn interacts the co-repressor N-CoR and with the histonedeacetylase mRPD1 (Heinzel, T., Lavinsky, R. M., Mullen, T.-M., S{hacekover (s)}derstr{hacek over (s)}m, M., Laherty, C. D., Torchia, J., Yang,W.-M., Brard, G., Ngo, S. D. & al., e. (1997) Nature 387, 43-46). Toexamine gene-specific activation, transcriptional activators aregenerated by fusing the zinc finger polypeptide to amino acids 413 to489 of the herpes simplex virus VP16 protein (Sadowski, I., Ma, J.,Triezenberg, S. & Ptashne, M. (1988) Nature 335, 563-564), or to anartificial tetrameric repeat of VP16's minimal activation domain,(Seipel, K., Georgiev, O. & Schaffner, W. (1992) EMBO J. 11, 4961-4968),termed VP64. The transcription regulating domains can be operativelylinked to a DNA binding domain at either the N— or C-terminus of thebinding domain.

A transcription regulating domain, when present, can be situated ateither the N— or C-terminal of a present polypeptide or adjacent to andbetween a DNA binding domain and a linker (see FIG. 2). A polypeptide ofthis invention can contain one or more transcription regulating domains.Where a plurality of transcription regulating domains are present, eachdomain can be the same or different. Similarly, a single polypeptide cancontain both repressor and activation domains. FIG. 2 shows an exemplarypolypeptides of this invention having two DNA binding domains and eithera single repressor or single activation domain or a combination of suchrepressor and activation domains.

III. Polynucleotides and Expression Vectors

The invention includes a nucleotide sequence encoding a zincfinger-nucleotide binding polypeptide. DNA sequences encoding the zincfinger-nucleotide binding polypeptides of the invention, includingnative, truncated, and expanded polypeptides, can be obtained by severalmethods. For example, the DNA can be isolated using hybridizationprocedures which are well known in the art. These include, but are notlimited to: (1) hybridization of probes to genomic or cDNA libraries todetect shared nucleotide sequences; (2) antibody screening of expressionlibraries to detect shared structural features; and (3) synthesis by thepolymerase chain reaction (PCR). RNA sequences of the invention can beobtained by methods known in the art (See, for example, CurrentProtocols in Molecular Biology, Ausubel, et al., Eds., 1989).

The development of specific DNA sequences encoding zincfinger-nucleotide binding polypeptides of the invention can be obtainedby: (1) isolation of a double-stranded DNA sequence from the genomicDNA; (2) chemical manufacture of a DNA sequence to provide the necessarycodons for the polypeptide of interest; and (3) in vitro synthesis of adouble-stranded DNA sequence by reverse transcription of mRNA isolatedfrom a eukaryotic donor cell. In the latter case, a double-stranded DNAcomplement of mRNA is eventually formed which is generally referred toas cDNA. Of these three methods for developing specific DNA sequencesfor use in recombinant procedures, the isolation of genomic DNA is theleast common. This is especially true when it is desirable to obtain themicrobial expression of mammalian polypeptides due to the presence ofintrons.

For obtaining zinc finger derived-DNA binding polypeptides, thesynthesis of DNA sequences is frequently the method of choice when theentire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the formation of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid-carrying cDNA libraries which arederived from reverse transcription of mRNA which is abundant in donorcells that have a high level of genetic expression. When used incombination with polymerase chain reaction technology, even rareexpression products can be clones. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucleic Acid Research 11:2325, 1983).

IV. Pharmaceutical Compositions

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of apolypeptide of this invention or a therapeutically effective amount of anucleotide sequence that encodes such a polypeptide in combination witha pharmaceutically acceptable carrier.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a human without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike which would be to a degree that would prohibit administration ofthe composition.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in theart. Typically such compositions are prepared as sterile injectableseither as liquid solutions or suspensions, aqueous or non-aqueous,however, solid forms suitable for solution, or suspensions, in liquidprior to use can also be prepared. The preparation can also beemulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, as well as pHbuffering agents and the like which enhance the effectiveness of theactive ingredient.

The therapeutic pharmaceutical composition of the present invention caninclude pharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, propylene glycol,polyethylene glycol and other solutes. Liquid compositions can alsocontain liquid phases in addition to and to the exclusion of water.Exemplary of such additional liquid phases are glycerin, vegetable oilssuch as cottonseed oil, organic esters such as ethyl oleate, andwater-oil emulsions.

V. Uses

In one embodiment, a method of the invention includes a process formodulating (inhibiting or suppressing) expression of a nucleotidesequence comprising a binding motif, which method includes the step ofcontacting the binding motif with an effective amount of a subjectpolypeptide that binds to the motif. The binding motif is preferablylocated in a transcriptional control region of the target gene. Atranscriptional control region is any region of a gene involved inregulating transcription. An exemplary such region is a promoter. In thecase where the nucleotide sequence is a promoter, the method includesinhibiting the transcriptional transactivation of a gene containing azinc finger-DNA binding motif. The term “inhibiting” refers to thesuppression of the level of activation of transcription of a structuralgene containing a zinc finger-nucleotide binding motif, for example. Inaddition, the gene transcription regulating polypeptide may bind a motifwithin a structural gene or within an RNA sequence.

The term “effective amount” includes that amount which results in thedeactivation of a previously activated promoter or that amount whichresults in the inactivation of a promoter containing a zincfinger-nucleotide binding motif, or that amount which blockstranscription of a structural gene or translation of RNA. The amount ofgene transcription regulating polypeptide required is that amountnecessary to either displace a native zinc finger-nucleotide bindingprotein in an existing protein/promoter complex, or that amountnecessary to compete with the native zinc finger-nucleotide bindingprotein to form a complex with the promoter itself. Similarly, theamount required to block a structural gene or RNA is that amount whichbinds to and blocks RNA polymerase from reading through on the gene orthat amount which inhibits translation, respectively. Preferably, themethod is performed intracellularly. By functionally inactivating apromoter or structural gene, transcription or translation is suppressed.Delivery of an effective amount of the inhibitory protein for binding toor “contacting” the cellular nucleotide sequence containing the zincfinger-nucleotide binding protein motif, can be accomplished by one ofthe mechanisms described herein, such as by retroviral vectors orliposomes, or other methods well known in the art.

The term “modulating” refers to the suppression, enhancement orinduction of a function. For example, the gene transcription regulatingpolypeptide of the invention may modulate a promoter sequence by bindingto a motif within the promoter, thereby enhancing or suppressingtranscription of a gene operatively linked to the promoter nucleotidesequence. Alternatively, modulation may include inhibition oftranscription of a gene where the gene transcription regulatingpolypeptide binds to the structural gene and blocks DNA dependent RNApolymerase from reading through the gene, thus inhibiting transcriptionof the gene. The structural gene may be a normal cellular gene or anoncogene, for example. Alternatively, modulation may include inhibitionof translation of a transcript.

The promoter region of a gene includes the regulatory elements thattypically lie 5′ to a structural gene. If a gene is to be activated,proteins known as transcription factors attach to the promoter region ofthe gene. This assembly resembles an “on switch” by enabling an enzymeto transcribe a second genetic segment from DNA to RNA. In most casesthe resulting RNA molecule serves as a template for synthesis of aspecific protein; sometimes RNA itself is the final product.

The promoter region may be a normal cellular promoter or, for example,an onco-promoter. An onco-promoter is generally a virus-derivedpromoter. For example, the long terminal repeat (LTR) of retroviruses isa promoter region which may be a target for a zinc finger bindingpolypeptide variant of the invention. Promoters from members of theLentivirus group, which include such pathogens as human T-celllymphotrophic virus (HTLV) 1 and 2, or human immunodeficiency virus(HIV) 1 or 2, are examples of viral promoter regions which may betargeted for transcriptional modulation by a polypeptide of theinvention.

The Examples that follow show the use of polypeptides of this inventionto alter expression of polynucleotides encoding particular geneproducts. The Examples are representative of particular embodiments ofthis invention and are not limiting of the specification and/or claimsin any way.

EXAMPLE 1 Transcription Factor Polypeptides

A series of transcription factor polypeptides that target specific geneswere made and used to alter expression of gene products. E2c and E2x aresix finger proteins that bind in the post-transcriptional andpre-translatorial region of the erbB2 gene, as fusion proteins with theeffector domains vp64 and SKD they regulate erbB2 expression in bothdirections [Beerli, R. et al: PNAS (1998), 95, 14628-14633; and Dreier,B. et al: J Biol Chem Aug. 3, 2001; 276(31):29466-78]. E3 and E3Y aresix finger proteins that bind in the post transcriptional andpre-translatorial region of the erbB3 gene, as fusion proteins with theeffector domains vp64 and SKD they regulate erbB3 expression in bothdirections [Beerli, R. et al: PNAS(1998), 95, 14628-14633; and Dreier,B. et al: J Biol Chem Aug. 3, 2001; 276(31):29466-78]. Exemplarypolypeptides are shown below together with the DNA target sequence forthat polypeptide. The six finger proteins were generated as describedelsewhere (Segal, D et al: PNAS (1999), 96,2758-2763). E2cJ15E3Y (SEQ IDNO:6) MAQAALEPGEKPYACPECGKSFSRKDSLVRHQRTHTGEKPYKCPECGKSFSQSGDLRRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYACPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSRSDKLVRHQRTHTGGGGSGGGGTGEKPYACPECGKSFSDKKDLTRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSQLLAHLRAHQRTHTGEKPYACPECGKSFSQSGDLRRHQRTHTGEKPYKCPECGKSFSRSDNLVRHQRTHTGEKPYKCPECGKSFSDPGALRVH QRTHTGKKTSGQAG DNATARGET SEQUENCE: (SEQ ID NO:7) E2c: GGG GCC GGA GCC GCA GTG; (SEQ IDNO:8) E3Y: ATC GAG GCA AGA GCC ACC E2xJ15E3 (SEQ ID NO:9)MAQAALEPGEKPYACPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSRSDHLAEHQRTHTGEKPYKCPECGKSFSDKKDLTRHQRTHTGEKPYACPECGKSFSQSSNLVRHQRTHTGEKPYKCPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSDKKDLTRHQRTHTHTGGGGSGGGGTGEKPYACPECGKSFSDPGALVRHQRTHTGEKPYKCPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYACPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSQSSHLVR HQRTHTGKKTSGQAG DNATARGET SEQUENCE: (SEQ ID NO:10) E3: GGA GCC GGA GCC GGA GTC; (SEQ IDNO:11) E2X: ACC GGA GAA ACC AGG GGA E3J15E2x (SEQ ID NO:12)MAQAALEPGEKPYACPECGKSFSDPGALVRHQRTHTGEKPYKCPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYACPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSQSSHLVRHQRTHTGGGGSGGGGTGEKPYACPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSRSDHLAEHQRTHTGEKPYKCPECGKSFSDKKDLTRHQRTHTGEKPYACPECGKSFSQSSNLVRHQRTHTGEKPYKCPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSDKKDLTRHQ RTHTGKKTSGQAG DNATARGET SEQUENCE: (SEQ ID NO:10) E3: GGA GCC GGA GCC GGA GTC; (SEQ IDNO:13) E2X: ACC GGA GAA ACC AGG GGA E2cJ15E3 (SEQ D NO:14)MAQAALEPGEKPYACPECGKSFSRKDSLVRHQRTHTGEKPYKCPECGKSFSQSGDLRRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYACPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSRSDKLVRHQRTHTGGGGSGGGGTGEKPYACPECGKSFSDPGALVRHQRTHTGEKPYKCPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYACPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSQSSHLVRHQ RTHTGKKTSGQAG DNATARGET SEQUENCE: (SEQ ID NO:7) E2c: GGG GCC GGA GCC GCA GTG; (SEQ IDNO:10) E3: GGA GCC GGA GCC GGA GTC E2xJ15E3y (SEQ ID NO:15)MAQAALEPGEKPYACPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSRSDHLAEHQRTHTGEKPYKCPECGKSFSDKKDLTRHQRTHTGEKPYACPECGKSFSQSSNLVRHQRTHTGEKPYKCPECGKSFSQSSHLVRHQRTHTGEKPYKCPECGKSFSDKKDLTRHQRTHTHTGGGGSGGGGTGEKPYACPECGKSFSDKKDLTRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSQLAHLRAHQRTHTGEKPYACPECGKSFSQSGDLRRHQRTHTGEKPYKCPECGKSFSRSDNLVRHQRTHTGEKPYKCPECGKSFSDPGALRV HQRTHTGKKTSGQAG DNATARGET SEQUENCE: (SEQ ID NO:13) E2X: ACC GGA GAA ACC AGG GGA; (SEQ IDNO:8) E3Y: ATC GAG GCA AGA GCC ACC

Various combinations of E2c, E2x, E3 and E3y (linked with short or longlinkers) were attached to either a repressor domain (SKD) or activationdomain (VP64). The charts below summarize such polypeptide.

Short Linker Constructs:J15 Effector domain Zif1 Zif2 SKD E2c E3 SKD E2cE3y SKD E2x E3y SKD E3 E2x

Zif1 Zif2 Effector domain E2c E3 Vp64 E2c E3y Vp64 E2x E3y Vp64 E3 E2xVp64

Long Linker Constructs: J30 Effector domain Zif1 Zif2 SKD E2c E3 SKD E2cE2x SKD E2c E3y SKD E3 E3y SKD E3 E2x SKD E2x E3y SKD E2x E3

Zif1 Zif2 Effector domain E2c E3 Vp64 E2c E3y Vp64 E2x E3y Vp64 E2x E3Vp64 E3 E2x Vp64

Instead of the regular canonical linker between Zif domains (TGEKP) thetwelve fingers contain longer linkers to connect the six fingerproteins. They were introduced by PCR using the forward primers J15F orJ30F and pMalseq Back as reverse primer. The PCR template can be aregular three or six finger in pMal (See Scheme 1, below):

Sequence Primer Short Linker J15: Sequence Range: 1 to 90           >SmaI         |    >XmaI|      |  |     |  |  10           20           30 TGA GCC CGG GGG CGG TGG CTC GGGCGG TGG ACT CGG GCC CCC GCC ACC GAG CCC GCC ACC   >BsrFI          >BglII      |               |    >AgeI       >XbaI |     |           |   |      |     40    |   |  50           60 TGG GACCGG TTC CTC TAG ATC TTC CTC CAC ACC CTG GCC AAG GAG ATC TAG AAG GAG CTG           70           80 CGG GGA GAA GCC CTA TGC TTG TCC GGA CCC CCTCTT CGG GAT ACG AAC AGG CCT  90 ATG (SEQ ID NO:16) TAC (SEQ ID NO:17)

Sequence Primer Long Linker J30F: Sequence Range: 1 to 99  >XmaI ||          10           20           30 CCC GGG TCC TCT GGT GGC GGT GGCTCG GGC GGG CCC AGG AGA CCA CCG CCA CCG AGC CCG           40           50           60 GGT GGT GGG GGT GGT TCC ACT GGCGGT GGC CCA CCA CCC CCA CCA AGG TGA CCG CCA CCG                   >AgeI                    |            70       |   80           90 TCG GGCGGT GGT GGG ACC GGT TCC TCT AGA AGC CCG CCA CCA CCC TGG CCA AGG AGA TCTTCT TCC TCC (SEQ ID NO:18) AGA AGG AGG (SEQ ID NO:19)

The PCR Product was cleaved with XmaI and SpeI and any original Zifprotein cleaved with AgeI and SpeI. The long linker containing zincfinger was then inserted between AgeI and SpeI. XmaI/AgeI formcompatible cohesive ends and both restriction sites (XmaI/AgeI)disappear during that cloning step. A new finger can be inserted bycutting this construct AgeI/SpeI and the original finger XmaI/SpeI. Thusthe resulting twelve finger has the same restriction sites as a sixfinger (Scheme 2). Consequently the assembly can be extended to nfingers, and can also combine three with six fingers etc.

EXAMPLE 2 Binding to Target DNA and Transcription Regulation

Polypeptides from Example 1 were tested for their binding to specificDNA target sequences and for their ability to alter transcription. Theresults are summarized below.

The 12 finger fusion proteins between e2c/e2x and e3/E3y are able toregulate both genes at once. This hypothesis was tested by transfectingthe different pMXSKD-12 finger and pMX12finger vp64 constructs in293-Gag-Pol cells and infecting A431 cells with the resulting virus.Three days after infection the cells were harvested and analyzed forerbB2 and erbB3 expression levels by FACS. This procedure was done asdescribed previously (Segal, D et al: PNAS(1999), 96,2758-2763). ELISAdata of raw extracts of e2cJ15/30E3 and e2cJ15/30e3y show that all fourconstructs bind their respective targets.

Seven different constructs have been analyzed for regulatory effects onerbB2 and erbB3. Down regulation of both erbB2 and erbB3 to basal levelswas observed for pMXSKDE2cJ15E3. Most efficient up regulation of botherbB2 and erbB3 was observed with the construct pMX E2cJ15E3vp64, pMXE2xJ15E3vp64 also affects both genes, as well as e2cE3yvp64.

pMXe2cJ15e3 repressed erbB2 and erbB3 down to basal levels. The otherthree constructs work worse, they just affect erbB3 expression withoutrepressing erbB2. This is true for both, e2x, which has lower affinityto its target (Km=15 nM), and the high affinity (Km=0.5 nM) e2ccontaining constructs. As e2x is once on the N-terminus, and once on theC-terminus, different positioning within the twelve finger does notimprove erbB2 repression. This is not a question of zinc finger proteinexpression levels, as estimated by GFP expression. pMXe2cJ15e3 is one ofthe weaker expressors and the most effective repressor.

Also for the activators, pMXe2cJ15e3vp64 showed the best effect byclearly activating erbB2 and erbB3. In contrast to the repressors,however, the two other constructs also activated both genes. ErbB3 seemsto be activated a bit stronger compared to erbB2.

Double targeting within one promoter could increase the overall weakactivation effect of zinc fingers. For that purpose pcDNAe2cJ15e2xvp64and pcDNASKDe2cJ15e2x were transiently transfected in Hela cells,together with the Luciferase reporter construct E2p. 36 fold repressionwas observed for SKDe2c compared to 8 fold repression for SKDe2cJ15e2x.For activation, 45 fold activation was observed for the twelve fingercompared to 78 fold activation by vp64e2c.

The twelve finger construct pMXe2cJ15CD144#5 does activate erbB2 but notCD144 in A431 cells. Two independent clones were tested and showed thesame effect. One clone was fully sequenced and just one aa of the lasthelix was unreadable or ambiguous. Overview of repressor domains testedwith erbB2 Repression of erbB2 (as measured in transient reporter assay,Repression Domain Beerli et al.(1998) SKD (entire KRAB-domain) >90%  SID (mSin3 interaction domain) 80% ERD 50% hCIR cloned, not tested none(ZIF alone) 30%

EXAMPLE 3 General Procedures

Construction of Zinc Finger Library and Selection via Phage Display

Construction of the zinc finger library was based on the C-7 protein(U.S. Pat. No. 6,140,081). Finger 3 recognizing the 5′-GCG-3′ subsitewas replaced by a domain binding to a 5′-GAT-3′ subsite via a PCRoverlap strategy using a primer coding for finger 3(5′-GAG-GAAGTTTGCCACCAGTGGCAACCTGGTGAGGCATACCAAAATC-3′)(SEQ ID NO:20)and a vector-specific primer (5′-GTAAAACGACGGCCAGTGCCAAGC-3′)(SEQ IDNO:21). Randomization of the zinc finger library by PCR overlapextension was essentially as is well known in the art. The library wasligated into the phagemid vector pComb3H. Growth and precipitation ofphage were performed using standard techniques. Binding reactions wereperformed in a volume of 500 ml of zinc buffer A (ZBA: 10 mM Tris, pH7.5, 90 mM KCl, 1 mM MgCl₂, 90 mM ZnCl2 ), 0.2% bovine serum albumin, 5mM dithiothreitol, 1% Blotto (Bio-Rad), 20 mg of double-stranded,sheared herring sperm DNA containing 100 ml of precipitated phage (10¹³colony-forming units). Phage were allowed to bind to non-biotinylatedcompetitor oligonucleotides for 1 h at 4° C. before the biotinylatedtarget oligonucleotide was added. Binding continued overnight at 4° C.After incubation with 50 ml of streptavidin-coated magnetic beads(Dynal; blocked with 5% Blotto in ZBA) for 1 h, beads were washed 10times with 500 ml of ZBA, 2% Tween 20, 5 mM dithiothreitol, and oncewith buffer containing no Tween. Elution of bound phage was performed byincubation in 25 ml of trypsin (10 mg/ml) in Tris-buffered saline for 30min at room temperature.

Hairpin competitor oligonucleotides had the sequence 5′-GGCCGCN′N′N′ATCGAGTTTTCTCGATNNNGCGGCC-3′ (SEQ ID NO:22), where NNN represents thefinger-2 subsite oligonucleotides and N′N′N′ its complementary bases.Target oligonucleotides were biotinylated and usually added at 72 nM inthe first three rounds of selection and then decreased to 36 and 18 nMin the sixth and last round. As competitor a 5′-TGG-3′ finger-2 subsiteoligonucleotide was used to compete with the parental clone. Anequimolar mixture of 15 finger-2 5′-ANN-3′ subsites, except for thetarget site and competitor mixtures of each finger-2 subsites of thetype 5′-CNN-3′, 5′-GNN-3′, and 5′-TNN-3′ were added in increasingamounts with each successive round of selection. Usually no specific5′-ANN-3′ competitor mix was added in the first round.

Multitarget Specificity Assay and Gel Mobility Shift Analysis—The zincfinger-coding sequence was subcloned from pComb3H into a modifiedbacterial expression vector pMal-c2 (New England Biolabs). Aftertransformation into XL1-Blue (Stratagene) the zincfinger-maltose-binding protein (MBP) fusions were expressed by additionof 1 nM isopropyl b-D-thiogalactoside (IPTG). Freeze/thaw extracts ofthese bacterial cultures were applied in 1:2 serial dilutions to 96-wellplates coated with streptavidin (Pierce) and were tested for DNA bindingspecificity against each of the 16 5′-GAT ANN GCG-3′ target sites.Enzyme-linked immunosorbent assay (ELISA) was performed. Afterincubation with a mouse anti-MBP antibody (Sigma, 1:1000), a goatanti-mouse antibody coupled with alkaline phosphatase (Sigma, 1:1000)was applied. Detection occurred by addition of alkaline phosphatasesubstrate (Sigma), and the A405 was determined by a microtiter platereader with SOFTMAX2.35 (Molecular Devices). Gel shift analysis wasperformed with purified protein (Protein Fusion and Purification System,New England Biolabs).

Site-Directed Mutagenesis of Finger 2—Finger-2 mutants were constructedby PCR. As PCR template the pMal vector encoding for C7.GAT was used.PCR products containing a mutagenized finger 2 and 5′-GAT-3′ finger 3were subcloned via NsiI and SpeI restriction sites in frame with finger1 of C7 (5′-GCG-3′) into a modified pMal-c2 vector (New EnglandBiolabs).

Construction of Polydactyl Zinc Finger Proteins—Three-finger proteinswere constructed by finger-2 stitchery using the SP1C framework. Theproteins generated in this work contained helices recognizing 5′-GNN-3′DNA sequences, as well as 5′-ANN-3′ and 5′-TAG-3′ helices. Six fingerproteins were assembled via compatible XmaI and BsrFI restriction sites.Analysis of DNA-binding properties were performed using freeze/thawextracts from IPTG-induced bacteria. For the analysis of the capabilityof these proteins to regulate gene expression, they were fused to theactivation domain VP64 or repression domain KRAB of Kox-1; VP64(tetrameric repeat of the herpes simplex virus VP16 minimal activationdomain) and subcloned into pcDNA3 (Invitrogen) or the retroviralpMX-IRES-GFP vector) internal ribosome-entry site (IRES) and greenfluorescent protein (GFP).

Transfection and Luciferase Assays—HeLa cells were used at a confluencyof 40-60%. Cells were transfected with 160 ng of reporter plasmid (pGL3;Promega) containing the promoter sequence with zinc finger-binding sitesand 40 ng of effector plasmid (zinc finger-effector domain fusions inpcDNA3) in 24-well plates. Cell extracts were prepared 48 h aftertransfection and measured with luciferase assay reagent (Promega) in aMicroLumat LB96P luminometer (EG & Berthold, Gaithersburg, Md.).

Retroviral Gene Targeting and Flow Cytometric—As primary antibody anErbB-1-specific mAb EGFR (Santa Cruz Biotechnology), ErbB-2-specific mAbFSP77 (gift from Nancy E. Hynes), and an ErbB-3-specific mAb SGP1(Oncogene Research Products) were used. Fluorescently labeled donkeyF(ab9)2 anti-mouse IgG was used as secondary antibody (JacksonImmunoResearch).

Bacterial Extracts of pMal-Fusion Proteins for ELISA Assays

The selected zinc finger proteins were cloned into the pMal vector (NewEngland Biolabs) for expression. The constructs were transferred intothe E. coli strain XL1-Blue by electroporation and streaked on LB platescontaining 503 g/ml carbenecillin. Four single colonies of each mutantwere inoculated into 3 ml of SB media containing 50 3 g/ml carbenecillinand 1% glycose. Cultures were grown overnight at 37° C. 1.2 ml of thecultures were transformed into 20 ml of fresh SB media containing 50 3g/ml carbenecillin, 0.2% glycose, 90 3 g/ml ZnCl₂ and grown at 37° C.for another 2 hours. IPTG was added to a final concentration of 0.3 mM.Incubation was continued for 2 hours. The cultures were centrifuged at4° C. for 5 minutes at 3500 rpm in a Beckman GPR centrifuge. Bacterialpellets were resuspended in 1.2 ml of Zinc Buffer A containing 5 mMfresh DTT. Protein extracts were isolated by freeze/thaw procedure usingdry ice/ethanol and warm water. This procedure was repeated 6 times.Samples were centrifuged at 4° C. for 5 minutes in an Eppendorfcentrifuge. The supernatant was transferred to a clean 1.5 ml centrifugetube and used for the ELISA assays.

ELISA Assays—Finger-2 variants of C7.GAT were subcloned into bacterialexpression vector as fusion with maltose-binding protein (MBP) andproteins were expressed by induction with 1 mM IPTG (proteins (p) aregiven the name of the finger-2 subsite against which they wereselected). Proteins were tested by enzyme-linked immunosorbent assay(ELISA) against each of the 16 finger-2 subsites of the type 5′-GAT CNNGCG-3′ to investigate their DNA-binding specificity.

In addition, the 5′-nucleotide recognition was analyzed by exposing zincfinger proteins to the specific target oligonucleotide and threesubsites which differed only in the 5′-nucleotide of the middle triplet.For example, pCAA was tested on 5′-AAA-3′, 5′-CAA-3′, 5′-GAA-3′, and5′-TAA-3′ subsites. Many of the tested 3-finger proteins showedexquisite DNA-binding specificity for the finger-2 subsite against theywere selected.

1. A non-naturally occurring transcription factor polypeptide comprising a plurality of DNA binding domains operatively linked with a flexible peptide linker having from 5 to 50 amino acid residue sequences.
 2. The polypeptide of claim 1 that contains 2 or 3 DNA binding domains.
 3. The polypeptide of claim 1 wherein the linker has from 5 to 30 amino acid residues.
 4. The polypeptide of claim 1 wherein the linker has from 5 to 15 amino acid residue sequences.
 5. The polypeptide of claim 1 further comprising a transcription regulating factor.
 6. The polypeptide of claim 5 wherein the transcription regulating factor represses transcription.
 7. The polypeptide of claim 5 wherein the transcription regulating factor activates transcription.
 8. The polypeptide of claim 5 wherein the transcription regulating factor is located at the N-terminus of the polypeptide.
 9. The polypeptide of claim 5 wherein the transcription regulating factor is located at the C-terminus of the polypeptide.
 10. The polypeptide of claim 1 further comprising a plurality of transcription regulating factors.
 11. The polypeptide of claim 1 wherein each DNA binding domain contains from 3 to 6 zinc finger DNA binding peptides.
 12. The polypeptide of claim 11 wherein each DNA binding domain contains 6 zinc finger DNA binding peptides.
 13. The polypeptide of claim 12 further comprising one or more transcription regulating domains.
 14. A polynucleotide that encodes the polypeptide of claim
 1. 15. An expression vector that contains the polynucleotide of claim
 14. 16. A cell transfected with the polynucleotide of claim
 14. 17. A process of altering expression of a nucleotide sequence containing a binding motif, comprising the step of contacting the binding motif with an effective amount of a polypeptide of claim 1 that binds to the motif.
 18. A process of simultaneously altering expression of a first nucleotide sequence containing a first binding motif and a second nucleotide sequence that contains a second binding motif, the process comprising the step of contacting the binding motifs with an effective amount of a polypeptide of claim 1 that binds to both the first and second binding motif. 